This invention relates to the detection of the presence or absence of specific restriction sites in specific nucleic acid sequences using restriction endonculease cleavage of an end-labeled oligonucleotide probe annealed to the sequence spanning the site(s). An example of the use of this detection system is a method and test kit wherein an oligonucleotide probe is used for the direct analysis of human .beta.-globin genes in the detection of sickle cell anemia.
In recent years the study of the molecular basis of hemoglobinopathies has made major advancements. Using sickle cell anemia and .beta.-thalassemia as the model systems, researchers have attempted to understand more fully the molecular basis for these genetic diseases and to develop direct means of prenatal diagnosis.
Prior to 1981, work in this area centered on the study of restriction fragment length polymorphisms (RFLP). Researchers found that cleavage of the nucleotide sequences using site-specific restriction endonucleases yielded DNA fragments of defined length. Kan and Dozy, PNAS (1978) 75:5631-5635 reported RFLP's produced by HpaI cleavage of human .beta.-globin genes, indicating a correlation between the 13.0 kb variant of the normal 7.6 kb fragment and the sickle cell mutation. This method of linkage analysis, though very useful, requires analysis of family members and even then is frequently unable to distinguish between normal and mutant genes. Because linkage analysis is based on the cosegregation of RFLP's associated with a specific disease within a family, the analysis is limited or inconclusive in cases where family studies are incomplete or unavailable.
In 1981, Geever, et al., PNAS (1981) 78:5081-5085 first reported a direct means for diagnosing sickle cell anemia which did not rely upon linkage analysis. This represented a major improvement in the diagnostic methods available since family studies would no longer be required in order to complete the analysis. Geever, et al. reported the use of a restriction endonuclease DdeI which recognizes the nucleotide base sequence "CTNAG" (where N is any base) for direct analysis of sickle cell anemia. The DdeI recognition site is abolished by the mutation (A to T) in the sickle cell allele of the .beta.-globin gene. The results of this research indicated that use of such a specific endonuclease will result in the formation of restriction fragments varied in length dependent upon whether the mutation causing sickle cell anemia is present. A detailed description of the methods used by Geever, et al. for this direct analysis for sickel cell anemia is found in U.S. Pat. No. 4,395,486 issued July 26, 1983.
An improvement in the method of Geever, et al, is described by Orkin, et al., N. Engl. J. Med. (1982) 307: 32-36. The method differs from Geever, et al. in the use of a restriction enzyme MstII which also cleaves normal DNA but not sickle cell DNA. MstII generates larger fragments than those obtained with DdeI, and use of MstII eliminates some of the limitations present in the Geever, et al. method. Specifically, the modification by Orkin, et al. allows for the direct analysis of the sickle cell gene without the necessity of identifying the small DNA fragments resulting from digestion with DdeI. Further, it enables direct analysis of cells obtained directly from uncultured amniotic fluid.
Additional improvements on the method of direct analysis of sickle cell anemia are described by Conner, et al., PNAS (1983) 80: 278-282. In this article, a general method for the diagnosis of any genetic disease which involves a point mutation in the DNA sequence is described. The model system tested is the .beta.-globin gene associated with sickle cell anemia; however, other similar methods (Nature (1983), 304: 230-234) have been used for prenatal diagnosis of .alpha.1-antitrypsin deficiency. The techniques as applied to both sickle cell anemia and .alpha.1-antitrypsin employ to use of a 19 base length oligodeoxyribonucleotide probe (19-mer). In the analysis of sickle cell anemia using the Conner et al. method, distinguishing between the normal .beta.-globin gene and the sickle cell mutant requires specific probes approximately 19 bases in length and containing the single point mutation. This method is especially useful in the detection of .alpha.1-antitrypsin deficiency, since such a deficiency cannot be readily diagnosed by RFLP due to lack of restriction enzymes that yield clinically informative polymorphic patterns.
A drawback to the Conner, et al. method is the limitation inherent in using a 19 base oligomer probe. The distinction between the allelic variants is based on the thermal stability of the duplex formed between the genomic DNA and the synthetic oligodeoxyribonucleotide (19-mer) probe. Oligodeoxyribonucleotides much larger than the 19 base oligomer will not be sufficiently destabilized by a single base mismatch and so this approach cannot utilize probes substantially longer than 19 bases. However, given the complexity of genomic DNA, a 19-mer probe will hybridize to many DNA sequences in addition to the specific .beta.-globin DNA sequence. This problem necessitates a gel electrophoretic step in which the .beta.-globin fragment produced by digestion with a restriction endonuclease is separated from all the other fragments which hybridize to the probe. This step is essential in physically separating the "signal" (.beta.-globin) from the "noise".
Once the fragments have been separated, the gel is dried followed by "in situ" hybridization of the probe to the electrophoretically separated fragments. Such manipulations of the gel are time consuming and present a technically difficult method of analysis.
As discussed, the references cited describe a direct method for the detection of genetic diseases using restriction fragment length polymorphisms (RFLP) or differential hybridization; however, they are limited in their applicability to routine clinical testing because of the complexity and sophistication required to carry out the analysis. The present invention overcomes these limitations by describing a fast yet sensitive method for detecting the presence or absence of specific polymorphic (as well as non-polymorphic) restriction sites. The method can be applied to genetic disorders capable of prenatal diagnosis such as sickle cell anemia or other genetic disorders where a polymorphic restriction site is clinically informative.